Figure 3
The metA element binds SAM. (a) In-line probing of 156 metA RNA from A. tumefaciens. 32P-labeled RNA (NR, no reaction) and products resulting from partial digestion with nuclease T1 (T1), partial digestion with alkali (-OH), and spontaneous cleavage during a 40 h incubation in the presence of varying of SAM concentrations (1 μM to 6 mM) were separated by polyacrylamide gel electrophoresis. Product bands corresponding to certain G residues (generated by T1 digestion) and full length 156 metA RNA (Pre) are labeled. (b) Sequence and secondary structure model for A. tumefaciens metA RNA. Sites of structural modulation for the 156 metA derived from in-line probing are circled with red, green and yellow representing reduced, increased, and constant scission in the presence of SAM, respectively. (c) Dependence of spontaneous cleavage in various regions of 156 metA on the concentration of SAM. Band intensities for the five regions (labeled 1-5) on the in-line probing gel in (a) were quantitated and normalized to the maximum modulation observed. Data from each of these sites corresponds to an apparent Kd of around 1 μM (producing half maximal modulation of cleavage) when plotted against the logarithm of the SAM concentration. Theoretical curves for single ligand binding at sites where cleavage increases (black) and decreases (gray) with a Kd of 1 μM are shown for comparison.